USP7/Maged1-mediated H2A monoubiquitination in the paraventricular thalamus: an epigenetic mechanism involved in cocaine use disorder

The risk of developing drug addiction is strongly influenced by the epigenetic landscape and chromatin remodeling. While histone modifications such as methylation and acetylation have been studied in the ventral tegmental area and nucleus accumbens (NAc), the role of H2A monoubiquitination remains unknown. Our investigations, initially focused on the scaffold protein melanoma-associated antigen D1 (Maged1), reveal that H2A monoubiquitination in the paraventricular thalamus (PVT) significantly contributes to cocaine-adaptive behaviors and transcriptional repression induced by cocaine. Chronic cocaine use increases H2A monoubiquitination, regulated by Maged1 and its partner USP7. Accordingly, Maged1 specific inactivation in thalamic Vglut2 neurons, or USP7 inhibition, blocks cocaine-evoked H2A monoubiquitination and cocaine locomotor sensitization. Additionally, genetic variations in MAGED1 and USP7 are linked to altered susceptibility to cocaine addiction and cocaine-associated symptoms in humans. These findings unveil an epigenetic modification in a non-canonical reward pathway of the brain and a potent marker of epigenetic risk factors for drug addiction in humans.


RNA-seq
To assess the proportion of Vglut2 neurons in the dissected thalamus, we performed fluorescenceactivated cell sorting (FACS) of Vglut2 neurons specifically expressing tdTomato, and we found that 93.1 % of sorted cells showed Vglut2 expression (Supplementary Fig. 6a).A principal component analysis separated the replicates of the microdissected thalamus data on the basis of mouse genotype and cocaine or saline treatment (Supplementary Fig. 6b).In control mice, repeated cocaine injection induced upregulated expression of 252 genes and downregulated expression of 452 genes compared to the expression of these genes after saline treatment (Fig. 3b and Supplementary Fig. 6c).
A gene ontology (GO) term enrichment analysis indicated that genes with cocaine-induced downregulated expression were significantly associated with microtubule regulation, axonal projection assembly and ion transport, while genes with cocaine-induced upregulated expression were assigned to supramolecular fiber (actin) organization and apoptosis (Supplementary Fig. 6d).Thus, cocaine-dependent downregulation and upregulation of gene expression seemed to affect different, yet related, biological processes.
Only 145 genes were similarly downregulated by cocaine treatment in mice with the Maged1-cKO and control genotypes, and 297 genes were specifically repressed in a cocaine-dependent manner only in the Maged1-cKO mice (Fig. 3b).Moreover, the expression of many genes was upregulated by cocaine in Maged1-cKO mice (494 genes), of which the expression of 383 (77.5 %) genes was not upregulated in control mice (Fig. 3b).Thus, Maged1 inactivation mainly led to an altered transcriptional downregulation in the thalamus after cocaine administration (Fig. 3b).

Cocaine-adaptive behaviors
To dissociate a global effect on locomotor activity rather than a specific effect on cocaine-induced sensitization of Maged1 in the PVT, we compared the locomotor activity on the first day of saline injection and the last day of cocaine injection (after reaching the ceiling level) in the rescue model, Maged1 KO, AAV-Maged1 (PVT), and in the cKO model Maged1 loxP , AAV-Cre (PVT).While there was no spontaneous locomotor activity difference between the inactivation or the reexpression of Maged1 in the PVT in these 2 models, the sensitization was significantly stronger in the rescue group as illustrated by an interaction effect between mice group (rescue versus deletion in the PVT) and treatment (saline/cocaine) (Supplementary Fig. 13).
The primary reinforcing properties of cocaine were evaluated in Vglut2Cre::Maged1 loxP mice and corresponding controls using operant self-administration.Mice were first trained in 5 sessions of fixed-ratio 1 (FR1) followed by 5 sessions of FR3 (Supplementary Fig. 3b).During FR1 and FR3 sessions; no statistical differences were obtained in active nose-pokes between genotypes (Supplementary Fig. 3a, two-way ANOVA repeated measures, genotype F1,14=1.130,n.s.).The number of nose-pokes on the inactive lever was similarly reduced in both genotypes (Supplementary Fig. 3c).No difference was observed in active nose-pokes and number of infusions during the last two or three days of FR1 and FR3, respectively (genotype x time, F9,126=1.529,n.s., two-way ANOVA repeated measures).Similarly, the percentage of mice reaching the criteria of operant conditioning learning was 66.7 % for WT mice and 75.0 % for Vglut2Cre::Maged1 loxP mice following FR1 and FR3 training (chi-squared test = 0.125, n.s.).Importantly, during the three consecutive days after the acquisition criteria of stability, discrimination and more than ten infusions, the number of active nose-poking responses was significantly higher in the Vglut2Cre::Maged1 loxP mice than in the control mice (Supplementary Fig. 3d).

Human data
In the clinical sample, we observed that the associations between MAGED1 and transition from first cocaine use to CUD and between USP7 SNPs and cocaine-induced aggression, respectively, were not confounded by sex in Cox regression models (Supplementary Tables 3 and 4).Not only these associations remained significant when sex was included as a potential confounder in the model, but sex was not associated with the clinical outcomes per se.We also investigated associations between biological sex and both cocaine-induced aggression and transition from first cocaine use to CUD using bivariate analyses, showing neither was significant (cocaine-induced aggression mean =1.39 in women and 1.38 in men, median = 0 and interquartile range = 0-3 in both sexes, Mann-Whitney test, P = 0.8707; transition from first cocaine use to CUD median = 12, interquartile range = 0-60 in women and 24 months, interquartile range = 0-84 in men, log-rank test Chi2 = 1.2, P = 0.2).Cocaine induced locomotor sensitization (20 mg/kg, ip, mean ± s.e.m., n = 9 (control, WT), n = 15 (control, KO), n = 16 (KO, re-exp), control, KO versus KO, re-exp using two-way ANOVA followed by Sidak's post-test, mixed-effects model, P = 0.0268; days, P<0.0001; interaction factor (genotype x days), P = 0.0046).b, scheme of the experiment for Amg-specific re-exp of Maged1 with stereotaxic injection of AAV CMV-Maged1-eGFP in Maged1-KO mice.In situ hybridization autoradiograms of Maged1 mRNA (scale bar: 1mm) and quantification of optical densities in the Amg (n = 9 (control, WT), n = 15 (control, KO), n = 5 (KO, re-exp), P< 0.0001 using Kruskal-Wallis test).Cocaine induced locomotor sensitization (20 mg/kg, ip, mean ± s.e.m., n = 9 (control, WT), n = 15 (control, KO), n = 5 (KO, re-exp), control KO versus re-exp KO using two-way ANOVA followed by Sidak's post-test, mixed-effects model, P = 0.0020; days, P<0.0001; interaction factor (genotype x days), P = 0.0733).
Supplementary Figure 6.Flow cytometry of the thalamus followed by transcriptomic and gene ontology analysis.a, Scheme of the RNA-seq experiment.Flow cytometry showed that 93.1 % of microdissected thalamic tissue consisted of Vglut2 cells (n = 6).b, Principal component analysis revealed four clusters referring to control and Vglut2Cre::Maged1 loxP (cKO) treated either with saline or cocaine during 5 consecutive days.c, Volcano plot showing the significantly differentially expressed genes between control saline and control cocaine mice.(452 downregulated and 252 upregulated genes, log2 fold change ≥ 0.5, false discovery rate (FDR) < 0.05).d, Gene Ontology analysis (biological process) showing categories with statistically significant enrichment of downregulated (top) and upregulated (bottom) genes following cocaine administration in control mice.

Supplementary
Supplementary Figure1.Multiplexed fluorescent in situ hybridization of Maged1, Vglut1 and Vglut2.Multiplexed fluorescent in situ hybridization of Maged1, Vglut1 and Vglut2 mRNAs.The first column shows a parasagittal slice of a control mouse.The third and fifth columns show a higher magnification focused on the thalamus and on the cortex, respectively.We observed that Maged1 is ubiquitously expressed among Vglut1 and Vglut2 cells.It is also expressed in other types of neurons and unmarked cells.The second column shows a Maged1-cKO specifically in Vglut2 expressing neurons.The fourth and sixth columns show a higher magnification focused on the thalamus and on the cortex, respectively.We observed an absence of expression of Maged1 in the thalamic nuclei that is Vglut2 positive, but a normal expression in the other regions, not expressing Vglut2 or expressing Vglut1.SupplementaryFigure 2. Maged1 is not necessary for cocaine-induced locomotor sensitization in glutamatergic telencephalic neurons.a, In situ hybridization autoradiograms showing Maged1 mRNA (scale bar: 500 µm) b, Quantification of the relative cortical optical densities (control, Maged1 loxP , n = 5; Emx1Cre:: Maged1 loxP mice, n = 5; Mann-Whitney test, P < 0.0001) c, Cocaine-induced locomotor sensitization (20 mg/kg, ip injection, mean ± s.e.m., control, Maged1 loxP , n = 8; Emx1Cre:: Maged1 loxP mice, n = 8; two-way ANOVA control versus EMX1Cre::Maged1 loxP , repeated measures, P = 0.9588; days, P < 0.0001; interaction factor (genotype x days), P = 0.9519).Supplementary Figure 3. Reinforcing properties of cocaine: cocaine-induced (10mg/kg, ip) conditioned place preference (CPP) and operant conditioning maintained by cocaine (0.5 mg/kg per infusion, iv) self-administration. a, CPP scored as the percentage of time spent in the drug-paired compartment (mean ± s.e.m., n = 8 for Vglut2cre::Maged1 loxP and n = 7 for control mice, two-way ANOVA, time factor, P = 0,0024, Sidak's post test, **P = 0,0028 for test versus pretest for control, Maged1 loxP mice and P = 0.4555 for Vglut2cre::Maged1 loxP mice).b, Number of active nose-pokes during the acquisition (fixed ratio 1 (FR1) and 3 (FR3)) of self-administration, two-way ANOVA repeated measures, genotype x time, F=1.700, n.s.).c, Number of inactive nose-pokes during the acquisition (fixed ratio 1 (FR1) and 3 (FR3)) of self-administration, two-way ANOVA repeated measures, genotype x time, F=0.2463, n.s.).d, Time course of the active nose-pokes during the three days of accomplishment of acquisition criteria (mean ± S.E.M; n = 12 (control, Maged1 loxP mice), n = 4 (Vglut2Cre::Maged1 loxP mice), two-way ANOVA repeated measures, genotype F=7.746, P < 0.05).cells, amygdala (Amg) inactivation, double inactivation in the Amg and PFC, vGluT1 cell inactivation and (2) diencephalic Maged1 inactivation (PVT and Vglut2) (two-way ANOVA followed by Sidak's post hoc test, groups, P < 0.0001; treatment, P < 0.0001; interaction factor, P < 0.0001).e, Scheme of a sagittal section of the mouse brain that represents the different regions with targeted Maged1 inactivation.f, Superimposed cocaine-induced locomotor sensitization experiment showing clear superimposition of full Maged1 KO mice with the Vglut2 mice and of the Vglut1 mice with the control mice.Supplementary Figure 5. Partial effect of Maged1 re-expression in glutamatergic Vglut1/2 nuclei.a, scheme of the experiment for PFC-specific re-expression (re-exp) of Maged1 with stereotaxic injection of AAV CMV-Maged1-eGFP in Maged1-KO mice.In situ hybridization autoradiograms of Maged1 mRNA (scale bar: 1mm) and quantification of optical densities in the PFC (n = 9 (control, WT), n = 11 (control, KO), n = 16 (KO, re-exp), P< 0.0001 using Kruskal-Wallis test).